#l5l:lilf¥it Bull. For. & For. Prod. Res. Inst. No. 343, 1987

Preparation and Culture of Protoplasts of some

Japanese Cultivated Mushrooms

By

Masatake OHMAsAm, Yasuhisa ABE'2>, Hisahiko FuRUKAWA's>

Minoru TANIGUCHIW and Hitoshi NEnAcs>

Summary : Protoplasts were prepared from mycelia of 12 mushrooms, Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes, Pholiota nameko, Grifola frondosa, Tricholoma matsutake and others. Mycelia of these mushrooms were cultured in liquid media, col­lected by filtration and treated with mixtures of commercial mycolytic enzymes. Among combinations of enzymes, enzyme systems containing a) Cellulase "Onozuka'' RS and ,8-Giucronidase, b) Cellulase "Onozuka" RS, Driselase and ,8-Glucronidase and c) Cellulase "Onozuka" RS, Driselase and Zymolyase 5000 were effective for protoplast formation. Dependence of the efficiency of protoplast formation on the culture period was examined for Lentinus edodes, Pholiota nameko, Grifola frondosa and Auricularia polytricha.

The efficiency was also found to be dependent on the strain of the same mushroom. In the course of the study, the enzyme system containing Novozym 234 and chitinase was found to be highly efficient for protoplast formation from mycelia of mushrooms of the genus Pleurotus. Using the strain, FMC 235, of Pleurotus ostreatus, the authors studied conditions for protoplast formation using this enzyme system, these are, pH of the enzyme solution, effect of osmotic stabilizers, time course of protoplast formation and the effect of media used for mycelial culture. Protoplast of Lentinus edodes and Pleurotus ostreatus could be cultured to form mycelia, and culture conditions were further studied. In the course of the study, the authors found that by starting from dikaryotic mycelia of Pleurotus ostreatus, they could get monokaryotic mycelia as well as dikaryotic mycelia after formation of protoplast from dikaryotic mycelia and culture. The authors conclude that this is a simple method of monokaryotization of dikaryotic mycelia.

Introduction

Protoplast is a cell whose envelope lacks a cell wall and is composed only of plasma

membrane. Protoplasts have been used in cell fusion of plants and fungi for their breedingm>.

In addition to cell fusion, protoplasts are useful in such aspects of genetics and breeding of

microorganisms as transformations> and artificial mutation4>5>. Protoplasts are becoming

more and more important also in studies of physiology of fungi as well as in genetics and

breedings>.

In recent years protoplasts have also been studied for mushrooms7>9>. But for main

cultivated mushrooms in Japan such as Lentinus edodes (Berk.) Sing., Pholiota nameko (T.

Ito) S. Ito et Imai, Grifola frondosa S. F. Gray and Pleurotus ostreatus (Fr.) Que!., protoplasts

have not been studied sufficiently. In this report we studied formation of protoplasts from

mycelia of 12 edible mushrooms including main cultivated mushrooms mentioned above, and

culture of Pleurotus ostreatus and Lentinus edodes. A part of this paper was presented

elsew herelOl.

Received August. 9, 1986 {¥ i\i-47 Forest Protection-47 (1) (2) (3) (4) (5) Forest Protection Division

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Materials and Methods

Origanisms

Strains of 12 mushrooms, that is Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes

(Fr.) Sing., Pholiota nameko, Grifola frondosa, Pleurotus cornucopiae (Pers.) Rolland, Hypsi­

zygus marmoreus (PK.) Bigelow, Tricholoma matsutake Ito et Imai, Auricularia polytricha

(Mont.) Sacc., Agaricus bisporus (Lange) Sing., Pleurotus salmoneo-stramineus Vassilieva,

Pleurotus cystidiosus 0. K. Miller, and two Pleurotus spp. were used. All the strains were

subcultured from stock cultures of Section of Mushroom of this institute (strains with FMC

number) and were maintained on potato-dextrose-agar (PDA) medium. Monokaryotic

mycelia obtained by single spore isolation from fruiting bodies of these stock cultures were

also used.

Prepararion of protoplasts from mycelia

Forty ml of a liquid media in a 100 ml Erlenmeyer flask containing sucrose (1%), malt

extracts (1%) and yeast extracts (0. 4%) (SMY medium) were inoculated with the mushroom

mycelia used in the experiment. After culturing at 25°C for 10-20 days depending on the

kind of mushroom, mycelia were fragmented in a homogenizer (Nippon Seiki Co., Ltd.) in

SMY medium at 12000 rpm for the time depending the kind of mushrooms. Except for the

examination of media, 5 ml of the medium containing the fragmented mycelia were re-ino­

culated to 40 ml of fresh SMY medium and cultured for 2-7 days at 25°C. Mycelia were

filtered by a nylonmesh and washed with a buffer solution containing 0. 5 M mannitol, 0. 05 M

maleic acid-NaOH, pH 5. 5 (buffer A).

Mycelia were resuspended in 3 ml of buffer A containing various combinations of enzymes

listed in Table 1, and incubated in a water bath at 25°C-30°C with reciprocal shaking (60

revolutions/min.). After incubation, remaining mycelia were removed by filtration through

Mylacloth (Calbiochem. Co., Ltd.). For tests of preparation of protoplasts, a number of

protoplasts present in the filtered solution were counted by a haemocytometer (Thoma

Table 1. Mycolytic enzymes used in this study.

Enzyme Source Supplier /Manufacturer

Cellulase "Onozuka" R-IO* Trichoderma viride Y akult Pharmaceutical

lndust. Co. Ltd. Cellulase "Onozuka" RS* Trichoderma viride Yakult Pharmaceutical

Indust. Co. Ltd. Chitinase Streptomyces griseus Sigma Chemical Co. Driselase Irpex lacteus Kyowa Hakko Kogyo Co.

Ltd.

{3-G 1 ucronidase Helix pomatia Sigma Chemical Co.

Macerozyme R-10 Rhizopus sp. Y akult Pharmaceutical

Indust. Co. Ltd.

Novozym 234 Trichoderma harzianum Novo Industri A/S Enzyme Division

Zymolyase 5000 Arthrobacter luteus Kirin Breweries Co. Ltd.

* Cellulase "Onozuka" R-10 and Cellulase "Onozuka" RS are abbreviated as "Cellulase R-10" and "Cellu­lase RS," respectively, in this paper.

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counting chamber). In each test, an average of three replications were shown. For other

tests, the filtered solution was centrifuged at 530Xg for five minutes and precipitated proto­

plasts were resuspended in the buffer A.

Protoplasts were purified as follows (when necessary). Protoplasts resuspended in buffer

A were layered on the 0. 7 M sucrose solution containing 0. 05 M maleic acid-NaOH, pH 5, 5,

in a centrifuge tube and centrifuged at 530Xg for five minutes. Protoplasts floating on the

upper surface of the sucrose layer were recovered and resuspended in 0. 7 M mannitol con­

taining 0. 05 M maleic acid-NaOH, pH 5. 5.

Culture of protoplasts

Protoplasts were prepared aseptically, washed once with buffer A and serially diluted

with the same buffer. The protoplast solution (0. 1 ml) was mixed with 10 ml of SMY or

GMY medium containing the osmotic stabilizer, additives, and agar or agarose, and cultured

in 9 em plastic Petri dishes sealed with parafilm, at 25"C. For the test of the effect of

osmotic stabilizers, 0. 5 M sucrose was replaced with other osmotic stabilizers. For liquid

cultures, 0. 1 ml of protoplast solution was mixed with the SMY or GMY medium containing

the osmotic stabilizer and additive but without agar.

Esterase isozymes

Esterase isozymes of mycelia of Pleurotus ostreatus cultured in the SMY medium for

three weeks were separated and detected as described previously11>.

Enzymes

Enzymes used for the preparation of protoplasts in the present study are listed in

Table 1.

Chemicals

Yeast extracts an nutrient broth were obtained from Oxoid Limited Co .. Malt extracts

were obtained from Kyokuto Seiyaku Ind. Co., Polypeptone was obtained from Wako Pure

Chemical Ind. Ltd., Potato-dextrose-agar was obtained from Nissui Seiyaku Co., Other chemi­

cals were obtained from Wako Pure Chemical Ind. Ltd. or Nakarai Chemicals, Ltd ..

Results

1. Isolation of Protoplast& from Mycelia of 12 Mushrooms

Efficiency of protoplast formation from mycelia depended firstly on the combination of

enzymes used for preparation. Several combinations of commercial .enzymes, except for

Novozym 234 and chitinase, listed in Table 1, were tested for the preparation of protoplasts

from Lentinus edodes, Pleurotus ostreatus, Plwliota nameko and Auricularia polytricha. Results

are shown in Tebles 2~5. As shown in these tables, one-enzyme systems were not generally

effective. In most cases, mixture of two or more commercial enzymes were effective. Enzyme

systems containing a) Cellulase RS and 13-Glucronidase or b) Cellulase RS, Driselase and

13-Glucronidase or c) Cellulase RS, Driselase and Zymolyase 5000 showed better results. For

a combination of enzymes, that is, for one enzyme system, the number of protoplasts formed

were dependent on the kind of mushrooms.

The dependence of the number of protoplasts formed from cultured mycelia on different

strains of the same mushroom, and on the culture period was tested using four strains each

of Lentinus edodes, and Plwliota nameko, three strains of Grifola frondosa, and a strain of

Auricularia polytricha. Results are shown in Figs. 1~4. The enzyme system used in this

Table 2. Number of protoplasts formed from mycelia of Lentinus edodes (Strain FMC 2) using several combinations of enzymes.

Combination of enzymes

Driselase 2%

Cellulase RS 2%

Macerozyme R-IO 2%

Zymolyase 5000 0. 2%

,s-Glucronidase 0. I ml/ml

Cellulase R-10 2% Driselase 2%+Cellulase RS 2%+Zymolyase 5000 o. 2%

Driselase 2%+Macerozyme R-IO 2%+Cellulase R-IO 2%

Driselase 2% + ,g-Glucronidase 0. I ml/ml

Cellulase RS 2% + ,g-Glucronidase o. I ml/ml

Driselase 2%+Cellulase RS 2%+13-Glucronidase 0. I ml/ml

Incubations were performed at 25°C for 5 hours.

Number of protoplasts per g fresh weight

0. 72X JOG

6.81X106

O.llXIOG

0.14XJOG

0. 17XJOG

O.OOXJOG

12. 27 X J06

I. 55 X JOG

0. 82X JO&

21. 93X JQ&

25. 31 X JOG

Table 3. Number of protoplasts formed from mycelia of Pleurotus ostreatus (Strain FMC 235) using several combinations of enzymes.

Combination of enzymes

Driselase I%

Cellulase RS I%

Macerozyme R-IO I%

Zymolyase 5000 0. I%

,g-Glucronidase 0. I ml/2 ml

Driselase 2%+Cellulase RS I%

Driselase 2%+Zymolyase 5000 0. I%

Driselase 2% + ,g-Glucronidase 0. I ml/2 rnl

Driselase 2% +Cellulase RS I%+ Zymolyase 5000 0. I%

Incubations were performed at 25°C for 5 hours.

Number of protoplasts per g fresh weight

0.07XJOG

17.43XJOG

0.94XJOG

O. 38X 10G

68.13X106

73.20XJOI

25.30X106 20.03 X J06

125.53 X JOG

Table 4. Number of protoplasts formed from mycelia of Pholiota nameko (Strain FMC 262) using several combinations of enzymes.

Combination of enzymes

Driselase 2%

Cellulase RS 2% Macerozyme 2%

Zymolyase 5000 0. 2%

Cellulase R-10 2% Driselase 2%+Cellulase RS 2%+Zymolyase 5000 0.2%

Driselase 2% + Macellozyme 2% +Cellulase R -10 2%

Cellulase RS 2% + ,g-Glucronidase o. 1 ml/ml

Incubations were performed at 25°C for 5 hours.

Number of protoplasts per g fresh weight

0. 83X JOG I. 08X JOG

0. 54 X JOG

0.71XJOG

0. 62X 106

6.97X106

3. 87X106

19.30X!06

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Table 5. Number of protoplasts formed from mycelia of Auricularia polytricha (Strain FMC 356) using several combinations of enzymes.

Combination of enzymes Number of protoplasts per g fresh weight

Driselase 2%

Cellulase RS 2%

Cellulase R-IO 2%

0, oox 106

2. 37X 106

O.OOX!06

Driselase 2% +Cellulase RS 2% + Zymolyase 5000 0. 2%

Driselase 2%+Macerozyme 2%+Cellulase R-IO 2%

Cellulase RS 2% + ,B-Glucronidase 0. I ml/ml

55. 73X106

I, 30X 106

395. 47 X 106

15,13XJ06 Cellulase RS 2% + Zymolyase 5000 0, 2%

Incubations were performed at 25°C for 5 hours.

'~ .s;;; 00

~ .s;;; X 10~ <f)

l!! U-

00

"

~ x10" M

~ ..<::

"' ~ LL

tlO

tl "' "' c. .8 0

0:: 0 t .0 E ::::J z

~, I ' I \

I ' X I \

' / ' 'I \ .,, ' I \

I \

Culture Period (days)

' 'b

Fig. 1. Effect of culture period on the formation of protoplasts from mycelia of different strains of Lentinus edodes. Incubations were performed at 25°C for 5 hours. FMC 2:0-0, FMC 8:0---0, FMC 29: x---x, FMC 45: x-x.

A ... ' I' ' I \

Fig. 2. Effect of culture period on the formation of protoplasts from mycelia of different strains of Pholiota nameko.

I \

J<-..., '\ I' .... '

........ ' .... ' .... ,,

Culture Period (days)

Incubations were performed at 25°C for 5 hours.

FMC 281:0--0, FMC 282:x---x, FMC 289:0---0, FMC 295:x---x.

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Fig. 3. Effect of culture period on the formation of pro· toplasts from mycelia of different strains of Grifola frondosa. Incubations were perfor· med at 25°C for 5 hours.

FMC 320:0-0, FMC32l:x---x, FMC 323:0---0.

X10' ~

-& 45.0

~ ..c:: VI e u.

Vl

tl "' c. 0 0 0:: 0 Vl .0 E :J z

Culture Period (days)

~

.<::: tlD

~ X106

..c: e l.i...

tlD

2 VI

"' c. 0 0 0:: 0

1i ~ 0 00~---~-...;;;::..::.~~===.:::..::=.llf-......J~---z

Culture Period (days)

Fig. 4. Effect of culture period on the formation of protoplasts from my­celia of a ,strain of Auricularia polytricha, FMC 356. Incubations were perfomed at 25•c for 5 hours.

experiment was Cellulase RS, 2%, Macerozyme R-10, 2%, Driselase, 2%, and Zymolyase 5000,

0. 2%. As seen in these figures, number of protoplasts is dependent on the strains used for

a mushroom. The effect of the culture period was also dependent on the strain. But pro­

toplast formation at 7 days was generally lower than those at 2 days and/or 4 days.

Formation of protop!asts from mycelia was also tested for strains of seven mushrooms

using enzyme system 1) Cellulase RS, 2%, Driselase, 2%, Macerozyme R-10, 2%, and Zymol­

yase 5000, 0. 2%, and 2) Cellulase R-10, 2%, Driselase, 2%, Macesozyme, 2%, and Zymolyase

5000, 0. 2%. Results are shown in Table 6. In , some experiments, formation of protoplasts

from mycelia of Tricholoma matsutake was ascertained (Fig. 5). We could not find any

effective enzyme system for Agaricus bisporus.

2. Highly efficient formation of protoplasts from mycelia of Pleurotus ostreatus.

In the process of the present study, a cell wall lytic enzyme, Novozym 234, became

available. So, authors tested this enzyme as well as chitinase, which was known to degrade

chitin, an important component of the cell wall of fungi. Results of the test for the prepa·

ration of protoplasts from mycelia of a strain of Pleurotus ostreatus (FMC 235) is shown in

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Table 6. Number of protoplasts formed from mycelia of several mushrooms using two enzyme systems.

Mushroom and strain

Pleurotus ostreatus FMC 235

FMC 236

FMC 237

FMC 238

FMC 239

FMC 240

FMC 241

FMC 242

FMC 243

FMC 244

FMC 245

FMC 246

Pleurotus cystidiosus FMC 256

Pleurotus salmoneo-stramineus FMC 252

Pleurotus sp. FMC 253

Pleurotus cornucopiae FMC 257

Hypsizygus marmoreus FMC 228

FMC 229

FMC 231

FMC 232

FMC 233

Flammulina veltipes FMC 223

FMC 224

Incubations were performed at 25°C for 5 hours.

Number of protoplasts per g fresh weight

Enzyme system I) Enzyme system 2)

131 X J06 29X 106

89 X 106 ?OX 106

89X 106 14X106

94 X 106 22X 106

61X!06 26X106

167X!06 26X106

23X 106 !9X106

!39X 106 34 X 106

59 X 106 12X10S

181 X106 !29X106

178X106 78X 106

!29Xl06 36X 106

75X 106 25X 106

38X 106 23X 106

53 X 106 16Xl06

16Xl06 6X106

159X106 68X10S

70X 106 42X10S

23X106 4X 106

!65X106 64Xl06

107 X 106 48X106

78X 106 45X 106

78X 106 56 X 106

Enzyme system 1) Cellulase RS 2%, Driselase 2%, Macerozyme R-10 2% and Zymolyase 5000 0,2% Enzyme system 2) Cellulase R-10 2%, Driselase 2%, Macerozyme R-10 2% and Zymolyase 5000 0. 2%

Fig. 5. Formation of protoplasts from mycelia of Tricholoma matsutake.

A : protruding protoplasm of mycelia formed in the process of formation of protoplasts.

B : protoplast released from mycelium,

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Fig. 6. Enzyme systems containing Novozym 234 showed better results and produced a high

number of protoplasts even at 2 hours incubation. Further tests were conducted on enzyme

system E (Novozym 234, 1%, chitinase, 0.1%). Protoplasts formed by these enzymes from

mycelia of Pleurotus ostreatus are shown in Fig. 7.

Because this enzyme system was very effective, authors further studied the various con·

ditions for protoplast formation from mycelia of Pleurotus ostreatus. Firstly, pH dependence

of protoplast formation was studied. Results are shown in Fig. 8. High yield was obtained

between pH 3. 5~5. 5 and further experiments were performed at pH 5. 5. The effect of

osmotic stabilizers on the protoplast formation is shown in Fig. 9. In further experiments,

authors used mannitol routinely. Fig. 10 shows the time course of protoplast formation for

mycelia grown at 30°C and 15°C. Mycelia grown at 30°C produced a higher number of

protoplasts in a shorter incubation time than from mycelia grown at l5°C. But mycelia

grown at 15°C produced a higher number of protoplasts at 3 hours. The number of pro·

toplasts formed from both mycelia grown at 15°C and those grown at 30°C decreased at 5

hours.

.<: 00

~ .<:: V1

!!! LJ..

"" -....,_

2 V1

"' a. 0 0 0: 0 ~

.D E "' z

X108

6.0 - -r-

5.0 -~ -

4.0 --

3.0

""" r-

2.0 - r-

1.0 - r-

0.0 _.... -. A B c D E F G

Combination of Enzymes

Fig. 6. Formation of protoplasts from mycelia of Pleurotus ostreatus (FMC 235) using several combinations of enzymes.

A : Cellulase RS 2% + Driselase 2% + Zymolyase 5000 0. 1%. B : ,8-Giucronidase 0. 1 ml/ml. C: ,8-Glucronidase 0.1 ml/ml+Cellulase RS 1%. D : Novozym 234 1%. E: Novozym 234 1%+Chitinase 0.1%, F: Novozym 234 1%+,8-Glucronidase 0.05ml/ml. G: Novozym 234 1%+Cellulase RS 1%.

For each combination of enzymes, incubations were performed at 30°C for 2 hours (shown by left bar) and 4 hours (shown by right bar).

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Fig. 7. Protoplasts of Pleurotus ostreatus prepared from mycelia using Novozym 234 and chitinase.

2 en

"' a. .8 0 a: 0 .... Q)

..0 E :J z Q) > ·.;::;

"' Qi 0::

7.5

pH of the Enzyme Solution

Fig. 8. PH dependance of protoplast formation from mycelia of Pleurotus ostreatus (FMC 235). Incubations were performed at 30°C for 2 hours.

"' 100.0 t: r-"' 0. 0 e """" "- r-0 ~ n E 50.0 :> ,_ z "' > ....- r-~ a; 0:

0.0 o:s: ':3g; -x o:s: ~6 ~~ :>

0-< co

"'" (jlg (jl[ "'"' " '->Q

2[ ?,:~ 2" 2f' (]1 (]1

-~ 2 2 (\)

Osmotic Stabilizer

Fig. 9. Effect of osmotic stabilizers on the protoplast formation from mycelia of Pleurotus ostreatus (FMC 235). Incubations were performed at 30°C for 2 hours.

Efficiency of protoplast formation was found to be dependent on the medium used for

culture of mycelia. Effect of media used for preparation of mycelia is shown in Table 7.

Using the method described here, we could prepare protoplasts very efficiently from

mycelia of Pleurotus ostreatus, P. salmoneo-stramineus, P. cystidiosus and two Pleurotus spp.

3. Culture of protoplasts

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..c: 00

~

<I)

t)

"' c. 0 b 0:

X107

30.0

0 15.0

~ E " z

•

Period of Incubation (hours)

Fig. 10. Time course of protop­last formation from my­celia of Pleurotus ostrea­tus (FMC 235) grown at 30°C and l5°C.

Incubations were performed at 30°C. for 2 hours.

: Mycelia grown at 30°C. : Mycelia grown at 15°C.

Table 7. Effect of the culture medium on the number of protoplasts formed from mycelia of Pleurotus ostreatus (FMC 235) grown in the medium.

MM + 1% Casamino acids MM+t% Polypeptone

MM+l% Nutrient broth SMY

Medium

t% Sucrose+ 1% Polypeptone+O. 4% Yeast extracts 1% Casamino acids+ I% Malt extracts+O. 4% Yeast extracts 1% Polypeptone+ 1% Malt extracts+O. 4% Yeast extracts 1% Nutrient broth+ 1% Malt extracts+ a. 4% Yeast extracts

1% Malt extracts+O. 4% Yeast extracts

Incubations were porformed at 30°C for 2 hours. MM : Diammonium Hydrogenphosphate 1. 5 g

Glucose 20g Potassium Dihydrogen Phosphate 0. 45 g di-Potassium Hydrogen Phosphate 1 g Magnesium Sulfate 0. 5 g Thiamine Hydrochloride 120 pg Distilled water 11

No. of protoplasts per g fresh weight

0,5XJ07

14, 3X!07

11. 8X!07

20, ox 107

20.1XI07

26, I XJ07

31, 9X!07

25.9X!07

44.6XJ07

Protoplasts could be cultured both in liquid media and in solid media. Protoplasts pre­

pared from mycelia of Pleurotus ostreatus (FMC 235) and Lentinus edodes (FMC 2) using the

enzyme system Cellulase RS. 2%, Driselase, 2%, Zymolyase 5000, 0. 2%, were first cultured in

the liquid media and then in solid media. Results are shown in Table 8 and Table 9. As

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Table 8. Culture of protoplasts obtained from mycelia of Pleurotus ostreatus (FMC 235)*

No. of colonies for protoplasts cultured

Osmotic stabilizers and additives in liquid media for*

2 hours I I day 2 days I 3 days I 4 days

Mannitol 0. 5 M 7 1I 6 7 5

MgS04 0.5M 3 23 27 62 103

KCl 0.3M 6 3 13 I5 7

Saccharose 0. 5 M 3 I3 29 41 41

MgS04 0. 5 M+O.I% NAG** 7 I7 55 I01 -MgS04 0. 5 M + 1% Polypeptone 10 10 23 141 80

* Protoplasts prepared using the enzyme system Cellulase RS 2% + Driselase 2% + Zymolyase 5000 0. 2%, were first cultured at 25°C for 2 hours-4 days in liquid media containing glucose 1%, malt extracts 1%, yeast extracts 0. 4% (GMY) and osmotic stabilizers and additives shown in the left column and then cultured at 25°C in solid media containing GMY, 0. 6 M mannitol and 0. 5% Agar. Number of colonies thus formed after 2 weeks of total culture period were counted.

** NAG: N-acetylglucosamine.

Table 9. Culture of protoplasts obtained from mycelia of Lentinus edodes (FMC 2)*

Osmotic stabilizers and additives

Mannitol 0. 5 M

MgS04 0.5M

KCl 0.3M

Saccharose o. 5 M

MgSO, 0.5M+I% NAG**

MgS04 0. 5 M +I% Polypeptone

*, ** ; See legends for Table 8.

No. of colonies for protoplasts cultured in liquid media for*

2 hours I 1 day 2 days 4 days 7 days

57

22

60

6I

29

22

38 90 73 31

II 2I 11 8

47 64 49 6

59 I03 83 4I

I2 11 0 4 IO 10 7 7

Fig. 11. Regeneration of a protoplast of Pleurotus ostreatus.

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seen in these tables, first culture in liquid media improved the efficiency of culture of pro­

toplasts for both mushrooms. But the effect of osmotic stabilizers and additives were

different for different mushrooms. Because N-acetylglucosamine improved the culture of

protoplasts of Pleurotus ostreatus, it was added to media in the following experiments. Ex­

amples of regenerating protoplasts and hyphae regenerated from protoplasts of Pleurotus

ostreatus in liquid media are shown in Figs. 11 and 12.

For protoplasts of Pleurotus ostreatus prepared using the enzyme system, Novozym 234+

chitinase, the effect of preculture in liquid media was not so clear. The effect of osmotic

stabilizers, however, was clearly observed. An example of the effect the above on the

culture of protoplasts of Pleurotus ostreatus in solid media is shown in Fig. 13. For Pleurotus

ostreatus, MgS04 was effective.

In the culture of protoplasts, pH of the medium had a serious effect on the culture

Fig. 13. Effect of osmotic stabilizers on the culture of protoplasts of Pleurotus ostreatus (FMC 235).

Protoplasts were prepared from mycelia of FMC 235 using Novozym 234 1%+chitinase 0.1%. They were washed and properly diluted in 0. 5 M man­nitol and then cultured at 25°C in solid media containing SMY, osmotic stabilizer, 0.1% N-acetyl­glucosamine and 0. 7% agarose. After 7 days, numbers of colonies were counted.

<J) til ·c: 0 0 u 0 >-til .0 E " z

100

50

0

Fig. 12. Hyphae of Pleurotus ostreatus regenerated from a protoplast.

c;s: -(f) as: -x -?< -(f) 00) ~~ On Oo

<0 . 0 Ol (Jl 0 N (na-(Jl(f) Olo (Jl:J

;:::§'. <f> 01 ~[ ~?

-::;-~CD ;:::,

~ -0 -Q. <f> CD

Osmotic Stabilizers

200

VI Q)

·c: 0 0 <..)

0 ~ Q) 100 .0 E ::J z

pH of the Medium

Fig. 14. Effect of pH of the medium on the culture of protoplasts of Pleurotus ostreatus (FMC 235). For culture of protoplasts, see the legend of Fig. 13.

• I I I

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t t Fig. 15. Monokaryotization of dikaryotic

mycelia of Pleurotus ostreatus, FMC 235, by formation and culture of protoplasts.

Esterase isozymes of mycelia which were obtained by culture of protoplasts prepared from dikaryotic mycelia of FMC 235, are shown here. An arrow of thick, solid line shows isozymes of the dikaryotic mycelia and arrows of narrow, solid and broken line show isozymes of two types of monokaryons.

efficiency. Protoplasts of FMC 235 were prepared and diluted as described in the legend of

Fig. 13 and cultured in media containing SMY, 0. 5 M MgS04, and maleic acid-NaOH buffer

to adjust pH. After 9 days of culture, the number of colonies were counted. Results are

shown in Fig. 14. As seen in this figure, pH near neutrality is good for the culture of pro·

toplasts of Pleurotus ostreatus in the pH range tested here.

In the course of experiments of preparation and culture of protoplasts of Pleurotus

ostreatus, we found that monokaryotic mycelia could also be obtained from the culture of

protoplasts. Colonies whose hyphae have no clamps could be recovered. They could be

mated with other monokaryotic .mycelia of other strains of Pleurotus ostreatus to produce

dikaryotic mycelia. Esterase isozymes were analyzed for these monokaryons, cultured from

protoplasts, as well as for dikaryons, cultured from protoplasts, and original stock. The

result is shown in Fig. 15. As seen from the figure, we could get two types of monokaryons.

Discussion

We were able to prepare protoplasts from mycelia of 12 species of mushrooms. Although

Moores> prepared protoplasts from Coprinus cinereus using only chitinase or Helicase and de

-168-

Vries et azm prepared protoplasts from a number of mushroom using only an enzyme prepared

from Trichoderma viride, our results indicated that generally mixed enzyme systems were

more effective. This fact seems to be related to the complex nature of the cell wall of mush­

room mycelials>. We could find effective enzyme systms for important cultivated mushrooms,

Lentinus edodes, Pleurotus ostreatus, Pholiota nameko and Auricularia polytricha. Also for Gri[ora

[rondosa we could get a fairly high number of protoplasts.

Figs. 1-4 and Table 6 clearly shows that the efficiency of an enzyme system is highly

dependent on the strain, even for the same mushroom. It is also dependent on the culture

period of mycelia. As shown in Figs. 1-4, seven-day cultures were generally ineffective for

protoplast formation compared with younger cultures. Table 7 shows protoplast formation

from Pleurotus ostreatus is also affected by the media used for culture of mycelia. It was

also observed that cultures containing large fractions of aerial hyphae were ineffective for

protoplast formation. These facts indicate that protoplast foramation from mycelia is severely

controlled by the physiological nature of mycelia.

We have established highly efficient enzyme systems for Pleurotus ostreatus as shown in

Fig. 6. The system seem to be superior to the method described by Yamada et el (1983)14>

for the preparation of protoplasts from mycelia of Pleurotus ostreatus. One of our enzyme

systems, Novozym 234+Chitinase, was effective not only for Pleurotus ostreatus but also for

other mushrooms of the genus Pleurotus.

Protoplasts of Pleurotus ostreatus and Lentinus edodes could be regenerated and cultivated.

Materials used for osmotic stabilizers had a serious effect on the efficiency of regeneration

and cultivation of protoplasts. But as seen in Table 8, 9 and Fig. 13, an effective osmotic

stabilizer was different for P. ostreatus and L. edodes. Regeneration of protoplasts of P. ostre­

atus were more effective at pH 6. 5 than at more acidic pH regions.

As described in "Results", dikaryotic mycelia of Pleurotus ostreatus could be easily mono­

karyotized by protoplast formation. Two types of monokaryon could be obtained as was evi­

denced by isozyme analysis. This technique seems to be more effective and easier than mono­

karyotization by culture in the special medium15l, Monokaryotization by protoplast formation

seems to have two advantages over the conventional method of getting monokaryotic mycelia

from isolated basidiospores.

1. We can get monokaryons easily and at any time.

2. We can always get monokaryons with a constant genetic nature in breeding.

Therefore monokaryotization by protoplast will be very useful in the practical breeding

of mushrooms.

References

1) CaALEFF, R. S.: Genetics of Higher Plants, Applications of Cell Culture, Cambridge

University Press, 184 P. P., (1981)

2) FERENczv, L. : Microbial Protoplast Fusion. In "Genetics as a Tool in Microbiology" ed.

by S. W. Glover and D. A. Hopwood. Cambridge University Press., p. 1-p. 34, (1981)

3) SANDRI·GoLoiN, R. M., A. L. GoLDIN, M. LEVINE and J, GLoRIOso: High-Efficiency Transfer

of DNA into Eukaryotic Cells by Protoplast Fusion. Methods in Enzymology, Academic

Press, 101, p. 402-p. 411, (1983)

4) KELLER, U. : Highly Efficient Mutagenesis of Claviceps purpurea by Using Protoplasts.

-169-

Appl. Environ. Microbial., 46, 580-584, (1983)

5) KIM, K. S., N.Y. CHo, H. S. PAI and D. D. Y. RYu: Mutagenesis of Micromonospora rosaria

by Using Protoplasts and Mycelial Fragments. Appl. Environ. Microbial., 46, 689-693,

(1983)

6) VILLANUEVA, J, R., I. GARCIA·AcHA, S. GAscoN and F. URusuRu: Yeast, Mould ane Plant Pro·

toplasts, Academic Press, 381 pp., (1973)

7) de VRIES, 0. M. H. and }. G. H. WEssELs: Release of Protoplasts from Schizophyllum com·

mune by a Lytic Enzyme Preparation from Trichoderma viriede. J. Gen. Microbial., 73, 13-

22, (1972)

8) MooRE, D. : Production of Coprinus Protoplasts by Use of Chitinase or Helicase. Trans.

Br. mycol Soc., 65, 134-136, (1975)

9) UsHIYAMA, R. and Y. NAKAI : Protoplasts of Shiitake, Lentinus edodes (Berk.) Sing Rept.

Tottori Mycol. Inst. (Japan), 15, 1-5, (1977)

10) 0HMASA, M., Y. AsE, H. FuRUKAwA, M. TANIGUCHI and H. NEDA: Preparation, Culture and

Fusion of Protoplasts of Mushrooms of the Genus Pleurotus. In "proceedings of the 28th

annual meeting of the Mycological Society of Japan", p. 59, (1984)

11) 0HMASA, M. and H. FURUKAWA :Analysis of esterase and malate dehydrogenase isozymes

of Lentinus edodes by isoelectric focusing for the identification and discrimination of stocks.

Trans. mycol. Soc. Japan, 27, 79-90, (1986)

12) de VRIES, 0. M. H. and J, G. H. WEsSELs: Effectiveness of a Lytic Enzyme Preparation

from Trichoderma viride in Releasing Spheroplasts from Fungi, Particularly Basidiomycetes.

Antonie van Leeuwenhoek, 39, 397-400, (1973)

13) FARKAs, V. :The Fungal Cell Wall. In "Fungal Protoplasts: Applications in Biochemistry

and Genetics" ed. by J. F. Peberdy and L. Ferenczy, Marcel Dekker, Inc., p. 3-29, (1985)

14) YAMADA, 0., Y. MAGAI, Y. KAsHIWAGI, T. SHIRATORI and T. SAsAKI: Formation and Regene·

ration of Flammulina velutipes and Pleurotus ostreatus Protoplasts. Nippon Shokuhin Kogyo

Gakkaishi, 30, 495-500, (1983)

15) LEAL-LARA, H. and G. EGER·HUMMEL: A Monokaryotization Method and its Use for Genetic

Studies in Wood-rotting Basidiomycetes. Theor. Appl. Genet., 61, 65-68, (1982)

-170- 林業試験場研究報告第 343 号

日本産栽培きのこのプロトプラストの調製と培養

大政正武ω. 阿部恭久叩・古川久彦〈別

谷口 実〈引・根田 仁(6)

摘要

プロトプラストは，細胞融合や形質転換をはじめとする，きのとではとれまで行われていなかった新し

い育種法を可能にするものとして，大きな期待が寄せられている。プロトプラストの作成と菌糸の再生に

ついてはとれまでスエヒロタケやヒトヨタケ属のきの ζなどについての研究はあるがが，シイタケ，ヒラ

タケ，ナメコ，マイタケなど日本産の栽培きの乙については研究が少なかった。本研究は，乙れらの日本

産の栽培きのとについて，プロトプラストの調製法と培養法を検討し，上記の新しい育種法を開拓するた

めの基礎的知見を得るととを目的として行ったものである。

日本産栽培きのと 12 種の菌糸からプロトプラストの調製を行った。 とれらにはシイタケ， ヒラタケ，

エノキタケ，ナメコ，マイタケ，マツタケ他が含まれる。きのとの菌糸を液体培養し，集菌後，菌の細胞

壁溶解酵素の処理でプロトプラストを得た。市販酵素の組み合せのうち， a)セ Jレラーゼ RS， ß-クツレクロ

ニダーゼ， b)セルラーゼ RS， ドリセラーゼ， βーグルクロニダーゼ， c) セJレラーゼ RS， ドリセラーゼ，

ザイモリアーゼ 5000 の酵素系が良い成績を示した。シイタケ， ナメコ，マイタケ，アラゲキクラゲにつ

いてプロトラプスト調製に対する菌糸の培養期間依存性を検討した。また系統の違いによってもプロトプ

ラスト調製は異なった。ノポザイム 234 とキチナーゼの組み合せはヒラタケ属のきのとに対してきわめて

良い成績を示したので，プロトプラスト調裂のさらに詳しい条件，すなわち， pH依存性，浸透圧調節剤

の影響，処理時間依存性，菌糸培養の培地の影響等を検討した。シイタケとヒラタケのプロトプラストは

培養を行なうととができたので，その条件をさらに検討した。その過程で，ヒラタケの 2 核菌糸からプロ

トプラストを得て培養すると 2 核菌糸と同時に 1 核菌糸が再生するととが見出され， 2 核菌糸の 1 核化の

簡便法として使えるととが分った。

1986年 8 月 9 日受理

(1) (2) (3) (4) (5) 保護部